Download Analysis of genetic systems using experimental evolution and whole

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project

Document related concepts

Artificial gene synthesis wikipedia , lookup

Minimal genome wikipedia , lookup

Dual inheritance theory wikipedia , lookup

History of genetic engineering wikipedia , lookup

Metagenomics wikipedia , lookup

Genome (book) wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Pathogenomics wikipedia , lookup

Human genetic variation wikipedia , lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup

Whole genome sequencing wikipedia , lookup

Genomic library wikipedia , lookup

Oncogenomics wikipedia , lookup

RNA-Seq wikipedia , lookup

Frameshift mutation wikipedia , lookup

Genomics wikipedia , lookup

Point mutation wikipedia , lookup

Epistasis wikipedia , lookup

Genome evolution wikipedia , lookup

Adaptive evolution in the human genome wikipedia , lookup

Mutation wikipedia , lookup

Population genetics wikipedia , lookup

Koinophilia wikipedia , lookup

Microevolution wikipedia , lookup

Transcript
Minireview
Analysis of genetic systems using experimental evolution and
whole-genome sequencing
Matthew Hegreness*† and Roy Kishony*‡
Addresses: *Department of Systems Biology, Harvard Medical School, Longwood Avenue, Boston, MA 02115, USA, †Department of
Organismic and Evolutionary Biology and ‡School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.
Correspondence: Roy Kishony. Email: [email protected]
Published: 1 February 2007
Genome Biology 2007, 8:201 (doi:10.1186/gb-2007-8-1-201)
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2007/8/1/201
© 2007 BioMed Central Ltd
Abstract
The application of whole-genome sequencing to the study of microbial evolution promises to
reveal the complex functional networks of mutations that underlie adaptation. A recent study of
parallel evolution in populations of Escherichia coli shows how adaptation involves both functional
changes to specific proteins as well as global changes in regulation.
The comparative study of extant genomes has revolutionized
biology, shedding light not only on evolution but also on
physiology, genetics and medicine. But the utility of
comparisons among naturally evolved isolates is lessened by
incomplete knowledge of the environment to which the
organisms adapted. Precise knowledge of conditions is
attainable only in comparative genomic studies of organisms
that have diverged under the controlled conditions of the
laboratory, where it is possible to run replicate experiments
that distinguish which outcomes are inevitable and which
the result of mere chance.
Advanced sequencing and mutation-detection technologies
now make it possible to reveal the complete genetic basis for
an adaptive trait that separates an evolved clone from a
reference strain [1-4]. The first whole-genome sequencing of
cellular organisms adapted to controlled laboratory conditions
has already revealed mutations that contribute to symbiosis
[1] and cooperative behavior [5-7]. A new study by Herring et
al. [8] takes whole-genome sequencing a significant step
further by exploring parallel evolution and its dynamics in
replicate populations of Escherichia coli. They also provide
direct characterizations of the effects of the detected
mutations using site-directed mutagenesis. Their results offer
clues to how complex biological systems function and evolve,
suggesting that adaptive regulation can occur not only at the
loci of genes that are directly involved in the adaptive trait but
also in distant areas of the network. Whole-genome
sequencing of parallel evolved strains promises to reveal novel
functional links among genes and genetic modules. Future
studies may be able to use genome-sequencing technologies to
answer a range of pressing questions in biology and evolution:
how biological networks are constructed, constrained, and
modified; how clonal interference shapes the outcomes of
evolution; and what is the complete spectrum of genetic
mutations available to selection.
The advantages of bacteria for experimental
evolution
In 1893, HL Russell, a bacteriologist at the University of
Wisconsin, enumerated some of the “evident advantages
that bacteria possess for experimental research in
evolutionary biology” [9]. These included how the “physical
and chemical environment [in which bacteria grow] can be
so rigidly controlled that the variability of conditions …is
practically excluded”, as well as how, by virtue of short
generation times, a “rapid successive transference of cultures to fresh media can secure the effect of an experiment
covering an immense number of generations within a
limited space of time” [9]. Russell’s ideas appear to have
remained unrealized for nearly a century, but the field of
experimental evolution finally emerged as a vibrant and
independent discipline towards the end of the twentieth
Genome Biology 2007, 8:201
201.2 Genome Biology 2007,
Volume 8, Issue 1, Article 210
Hegreness and Kishony
century [10]. With advances in the culture and genetic
manipulation of microbes, investigators in the 1980s began
to let microbes compete and evolve in the laboratory. Early
studies used the ability to obtain precise fitness measurements in chemostats to reveal subtle fitness differences
associated with natural, induced, and engineered mutations
[11], demonstrating the direct link between metabolic flux
and fitness [12]. Later experiments, using long-term laboratory evolution of parallel lines, were aimed at the key
evolutionary question of how much variability we would
expect were we to replay the ‘tape of life’ [13]; that is, how
reproducible are evolutionary outcomes. The most
celebrated long-term parallel experiment was begun by RE
Lenski in 1988 with 12 replicate populations of Escherichia
coli and has been running continuously for almost 20 years
and more than 40,000 generations in glucose-limited
medium [14]. These long-term lines have shed much light on
the inherent variability of the evolutionary process at a range
of phenotypic levels [14,15]. Now, with recent advances in
genomic technologies, these questions have begun to be
addressed at the genotypic level [14,16-18]. With wholegenome sequencing, all genetic changes underlying an
adaptive trait can be revealed and their dynamics tracked
over time. The new study by Herring et al. [8] suggests some
of the ways in which whole-genome sequencing will provide
deeper insight into the connections between parallel
evolution at the genotypic and phenotypic levels.
Parallel adaptation in functional modules
One salient finding that has emerged from laboratory studies
of evolving microbes is that parallel evolutionary changes
are often seen in replicate populations adapting to a novel
environment. Parallel evolution is a hallmark of natural
selection: identical or very similar changes reach high
frequency or fixation in independent lineages evolving under
identical conditions. The use of parallel evolution to infer
that adaptation had occurred was first applied to morphological traits [19], but it has been even more convincing in
the world of molecules [4,14,20-25]. With their spartan
genomes, RNA and DNA viruses were the first organisms for
which individual genomes from replicate laboratory
populations were fully sequenced. Although whole-genome
sequencing reveals all the mutations between an evolved
strain and its ancestor, further experimentation is needed to
show whether any of these mutations are neutral and how
the various mutations combine to form an adaptive trait. In
addition to the revelation that the vast majority of mutations
that reach appreciable frequency in viral populations are
beneficial, such sequencing studies produced striking
examples of parallel evolution - often exactly the same
change in the same amino acid [26].
It is perhaps not so surprising that we should find a limited
set of changes and pervasive parallel evolution in viruses,
whose genomes are very small and which lack the
http://genomebiology.com/2007/8/1/210
complicated regulatory networks of the higher forms of life.
Evolution acts on biological function, and in viruses functions
are often mapped to single genes. In complex cellular life
forms such as bacteria and yeast, however, complex functions
are typically attributed to modules of genes [27]. We might
expect, therefore, that parallel evolution for cellular life does
not necessarily mean similar changes in the same genes but
rather similar changes in related modules. For example,
recent studies have found that a phenotype under significant
positive selection in Lenski’s long-term lines is the degree of
DNA supercoiling [21]. Although a candidate-gene approach
revealed the genes responsible for the changes in supercoiling
in some of the evolving strains, the genetic causes underlying
the same phenotype in many of his other strains remain
obscure [21]. Whole-genome sequencing of these lines could
undoubtedly reveal the many different genetic changes that
can produce the same parallel phenotypic change in DNA
topology, and it could thus unmask the supercoiling genemodule under selection. Through the revelation of parallel
cellular phenotypes produced by seemingly dissimilar genetic
changes, functional connections within and between genetic
modules [28-29] can now be revealed by experimental
evolution coupled with whole-genome sequencing.
The current study by Herring et al. [8] focuses on
metabolism and its regulation. Metabolism provides perhaps
the best example of a large cellular network (comprising
hundreds of genes) that is relatively amenable to quantitative phenotypic predictions at the whole-cell level [30-33].
Although the overall optimal fitness of adapting populations
limited by a given single metabolic resource can be predicted
[34,35], only some of the mutations underlying the actual
fitness changes appear in the list of candidate genes [34].
Presumably, a regulatory change in a remote location of the
network can have far-reaching and unexpected effects.
Using a new microarray-based technology of whole-genome
resequencing for identifying the changes between a known and
reference strain, Herring et al. [8] explore the parallel changes
in metabolic and regulatory networks that appeared in five
replicate E. coli populations that evolved separately in glycerol
minimal media for 44 days. This study provides new examples
of parallel evolution in candidate genes, but also, as a
consequence of the comprehensive sequence information
obtained, begins to provide examples of how remote changes
might propagate through complex networks and how seemingly
disparate changes can have similar phenotypic effects.
Herring et al. [8] observed parallel changes in both global
regulation patterns and local protein sequences. Resequencing
five clones - one clone from each of the replicate populations
- the authors identified 13 mutations. A single gene, glpK
(encoding glycerol kinase), was mutated in all five lineages.
The protein synthesized by this gene catalyzes the first step
in glycerol catabolism, and the mutations in this gene led to
more than 50% increases in the reaction rate of glycerol kinase.
Genome Biology 2007, 8:201
http://genomebiology.com/2007/8/1/201
Genome Biology 2007,
This is an exceptional example of parallel evolution that
resonates with the results from experimental viral evolution.
Apart from the glycerol kinase mutations, the most significant
mutations identified affected global transcription patterns.
The largest fitness changes (representing roughly half of the
total increase in growth rate) in any of the five populations
resulted from mutations in genes encoding the two major
subunits of RNA polymerase (rpoB and rpoC). In three of the
five populations, natural selection fixed a mutant variant of
rpoB or rpoC within 25 days from the start of the experiment.
The reason that these changes were beneficial is unknown.
Two of these populations subsequently experienced a sweep of
secondary mutations that were only conditionally beneficial;
these may represent compensatory changes that might have
been needed to alleviate the presumed deleterious effects of
global changes in gene expression. One of the populations that
did not have mutations in RNA polymerase had an 82 basepair deletion adjacent to crr, which encodes critical
component in catabolite repression. Herring et al. [8] suggest
that attenuation of crr expression, as well as the mutations in
the genes encoding RNA polymerase, may reduce the
expression of genes that lead to catabolite repression, which
inhibits growth on glycerol. The basis of these effects is still to
be identified.
Whole-genome sequencing coupled with the careful control
of conditions that is possible in laboratory evolution thus
allowed Herring et al. [8] to demonstrate how molecular
evolution proceeds both in cis and in trans: that is,
adaptation involves local changes to specific proteins (for
example, glpK) as well as remote regulatory changes.
Studying the basis of clonal interference by
whole-genome sequencing
Herring et al. [8] sequenced clonal samples from their
populations after 44 days. Sequencing of many clones from
each population is still technologically unfeasible. How
different would the results have been if it was possible to
sequence many different individuals from each evolving
population? Bacterial populations invariably show some
degree of genetic variability as a result of spontaneous
mutation rates and genetic drift of neutral and deleterious
alleles. But beneficial mutations are particularly important to
population heterogeneity, especially on laboratory timescales.
Microbial evolution invariably includes clonal interference
among competing lineages, that is, multiple distinct beneficial
mutations spread through a population at a given time [25,3645]. On laboratory timescales during which horizontal transfer
of mutations is negligible, beneficial mutations remain linked
to the genome in which they appeared, and so the spread of
one beneficial mutation can impede the spread of others.
Herring et al. [8] recognized that clonal interference shaped
the evolution of their populations, and they attempted to
Volume 8, Issue 1, Article 201
Hegreness and Kishony 201.3
discover competing clonal lineages by sequencing the handful of candidate genes suggested by their whole-genome
sequencing in search of alternative alleles. They found four
alternative glpK alleles in two populations. Furthermore,
their time course of allele frequency measurements shows
several telltale signs of clonal interference, such as transient
or permanent decreases in frequency of particular
beneficial alleles after an initial rise, indicating competition
with a fitter lineage. The independent appearances of
mutations in glpK and rpoC in replicate populations is a
less obvious consequence of clonal interference - many
beneficial mutations of small effect are probably spreading
through the population but do not reach high frequency by
the time the strong mutations in glpK and rpoC spread
through the population.
As whole-genome sequencing becomes cheaper and more
reliable, it will be easier to study clonal interference as a
mechanism affecting the overall rate of adaptation. One
question is whether clonal interference happens most
frequently between clones with roughly the same
phenotype - that is, competition between different
genotypic changes in the same specific genes, pathways, or
regulatory networks - or whether different phenotypic
changes are competing instead.
The raw material of evolution
In the relatively brief evolutionary timescales and moderate
population sizes of studies such as that of Herring et al. [8],
neutral mutations would not have had time to spread
appreciably in the population by genetic drift. Furthermore,
although new neutral and deleterious mutations would arise
every generation, deleterious and neutral alleles are pushed
toward extinction as lineages carrying strongly beneficial
alleles spread to fixation. Thus, it is not surprising that the
few mutations discovered by Herring et al. [8] were all
beneficial, and typically of large effect. In addition to
studying adaptive mutations, whole-genome sequencing
could be used to explore better the actual underlying
genotypic spectrum of mutations before selection’s
winnowing; that is, it could be used to look at what the raw
material presented to natural selection is and how it varies
across organisms and environments.
Whole-genome sequencing can elucidate the nature of
spontaneous mutations when coupled with experimental
evolution in mutation accumulation (MA) lines. MA lines are
evolved for many generations with as little selection as
experimentally feasible [46-49]. This is typically achieved by
putting a population through consecutive one-organism
bottlenecks every few generations. This allows one to
observe how deleterious, neutral and beneficial mutations
accumulate without selection. Whole-genome sequencing of
MA lines offers considerable promise for seeing the types of
mutations that arise in such selection-less experiments. This
Genome Biology 2007, 8:201
201.4 Genome Biology 2007,
Volume 8, Issue 1, Article 210
Hegreness and Kishony
will enable geneticists to go from the most basal level, the
mutations that compose the raw material for evolution, all
the way through gene function and regulation to the ultimate
evolutionary phenotype - fitness.
When we see how much whole-genome sequencing has
revealed about evolution in nature [50,51], we can imagine
how much more can be learned about evolution on a
laboratory timescale. By sequencing clones from populations
that have evolved in identical laboratory conditions, Herring
et al. [8] provide further evidence for the ubiquity of parallel
evolution on the genotypic level, and their study suggests that
remote changes are propagated through genetic systems.
Experimental evolution, coupled with genomic technologies,
is poised to answer many important questions at the interface
between cellular processes and observed evolutionary
consequences. Evolution is becoming a powerful tool for
studying biological processes, principles and systems.
Acknowledgements
For helpful comments and suggestions we would like to thank Alexander
DeLuna, Daniel Hartl, Ayellet Segré, Daniel Segré, Noam Shoresh,
Rebecca Ward and Pamela Yeh.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Shendure J, Porreca GJ, Reppas NB, Lin X, McCutcheon JP, Rosenbaum AM, Wang MD, Zhang K, Mitra RD, Church GM: Accurate
multiplex polony sequencing of an evolved bacterial
genome. Science 2005, 309:1728-1732.
Margulies M, Egholm M, Altman WE, Attiya S, Bader JS, Bemben LA,
Berka J, Braverman MS, Chen YJ, Chen ZT, et al.: Genome
sequencing in microfabricated high-density picolitre reactors. Nature 2005, 437:376-380.
Braslavsky I, Hebert B, Kartalov E, Quake SR: Sequence information can be obtained from single DNA molecules. Proc Natl
Acad Sci USA 2003, 100:3960-3964.
Segre AV, Murray AW, Leu JY: High-resolution mutation
mapping reveals parallel experimental evolution in yeast.
PLoS Biol 2006, 4:1372-1385.
Velicer GJ, Raddatz G, Keller H, Deiss S, Lanz C, Dinkelacker I,
Schuster SC: Comprehensive mutation identification in an
evolved bacterial cooperator and its cheating ancestor. Proc
Natl Acad Sci USA 2006, 103:8107-8112.
Fiegna F, Yu YTN, Kadam SV, Velicer GJ: Evolution of an obligate social cheater to a superior cooperator. Nature 2006,
441:310-314.
Foster KR: Sociobiology: the phoenix effect. Nature 2006, 441:
291-292.
Herring CD, Raghunathan A, Honisch C, Patel T, Applebee MK, Joyce
AR, Albert TJ, Blattner FR, van den Boom D, Cantor CR, et al.: Comparative genome sequencing of Escherichia coli allows observation of bacterial evolution on a laboratory timescale. Nat
Genet 2006, 38:1406-1412.
Russell HL: Bacteriology in its general relation (continued).
Am Nat 1893, 27:1050-1065.
Elena SF, Lenski RE: Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation. Nat Rev
Genet 2003, 4:457-469.
Dykhuizen DE, Hartl DL: Selection in chemostats. Microbiol Rev
1983, 47:150-168.
Dykhuizen DE, Dean AM, Hartl DL: Metabolic flux and fitness.
Genetics 1987, 115:25-31.
Gould SJ: Wonderful Life: The Burgess Shale and the Nature of History.
New York: WW Norton; 1990.
Lenski RE: Phenotypic and genomic evolution during a 20,000generation experiment with the bacterium Escherichia coli.
Plant Breeding Rev 2004, 24:225-265.
http://genomebiology.com/2007/8/1/210
15. Pelosi L, Kuhn L, Guetta D, Garin J, Geiselmann J, Lenski RE, Schneider D: Parallel changes in global protein profiles during longterm experimental evolution in Escherichia coli. Genetics 2006,
173:1851-1869.
16. Schneider D, Duperchy E, Coursange E, Lenski RE, Blot M: Longterm experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and
rearrangements. Genetics 2000, 156:477-488.
17. Papadopoulos D, Schneider D, Meier-Eiss J, Arber W, Lenski RE, Blot
M: Genomic evolution during a 10,000-generation experiment with bacteria. Proc Natl Acad Sci USA 1999, 96:3807-3812.
18. Lenski RE, Winkworth CL, Riley MA: Rates of DNA sequence
evolution in experimental populations of Escherichia coli
during 20,000 generations. J Mol Evol 2003, 56:498-508.
19. Simpson GG: The Major Features of Evolution. New York: Columbia
University Press; 1953.
20. Cooper TF, Rozen DE, Lenski RE: Parallel changes in gene
expression after 20,000 generations of evolution in
Escherichia coli. Proc Natl Acad Sci USA 2003, 100:1072-1077.
21. Crozat E, Philippe N, Lenski RE, Geiselmann J, Schneider D: Longterm experimental evolution in Escherichia coli. XII. DNA
topology as a key target of selection. Genetics 2005, 169:523-532.
22. Woods R, Schneider D, Winkworth CL, Riley MA, Lenski RE: Tests
of parallel molecular evolution in a long-term experiment
with Escherichia coli. Proc Natl Acad Sci USA 2006, 103:9107-9112.
23. Honisch C, Raghunathan A, Cantor CR, Palsson BO, van den Boom
D: High-throughput mutation detection underlying adaptive evolution of Escherichia coli-K12. Genome Res 2004,
14:2495-2502.
24. Ferea TL, Botstein D, Brown PO, Rosenzweig RF: Systematic
changes in gene expression patterns following adaptive evolution in yeast. Proc Natl Acad Sci USA 1999, 96:9721-9726.
25. Notley-McRobb L, Ferenci T: Adaptive mgl-regulatory mutations and genetic diversity evolving in glucose-limited
Escherichia coli populations. Environ Microbiol 1999, 1:33-43.
26. Wichman HA, Badgett MR, Scott LA, Boulianne CM, Bull JJ: Different trajectories of parallel evolution during viral adaptation.
Science 1999, 285:422-424.
27. Hartwell LH, Hopfield JJ, Leibler S, Murray AW: From molecular
to modular cell biology. Nature 1999, 402:C47-C52.
28. Segré D, DeLuna A, Church GM, Kishony R: Modular epistasis in
yeast metabolism Nat Genet 2005, 37:77-83.
29. Yeh P, Tschumi AI, Kishony R: Functional classification of drugs
by properties of their pairwise interactions. Nat Genet 2006,
38:489-494.
30. Kacser H, Burns JA: The control of flux. Symp Soc Exp Biol 1973,
27:65-104.
31. Heinrich R, Schuster S: The Regulation of Cellular Systems. Dordrecht:
Chapman & Hall; 1996.
32. Kauffman KJ, Prakash P, Edwards JS: Advances in flux balance
analysis. Curr Opin Biotechnol 2003, 14:491-496.
33. Varma A, Palsson BO: Stoichiometric flux balance models
quantitatively predict growth and metabolic by-product
secretion in wild-type Escherichia coli W3110. Appl Environ
Microbiol 1994, 60:3724-3731.
34. Dekel E, Alon U: Optimality and evolutionary tuning of the
expression level of a protein. Nature 2005, 436:588-592.
35. Fong SS, Palsson BO: Metabolic gene-deletion strains of
Escherichia coli evolve to computationally predicted growth
phenotypes. Nat Genet 2004, 36:1056-1058.
36. Gerrish PJ, Lenski RE: The fate of competing beneficial mutations in an asexual population. Genetica 1998, 102-103:127-144.
37. Muller HJ: Some genetic aspects of sex. Am Nat 1932, 66:118-138.
38. Crow JF, Kimura M: Evolution in sexual and asexual populations. Am Nat 1965, 99:439-450.
39. Lenski RE, Rose MR, Simpson SC, Tadler SC: Long-term experimental evolution in Escherichia coli. 1. Adaptation and divergence during 2,000 generations. Am Nat 1991, 138:1315-1341.
40. Miralles R, Gerrish PJ, Moya A, Elena SF: Clonal interference and
the evolution of RNA viruses. Science 1999, 285:1745-1747.
41. Arjan JA, Visser M, Zeyl CW, Gerrish PJ, Blanchard JL, Lenski RE:
Diminishing returns from mutation supply rate in asexual
populations. Science 1999, 283:404-406.
42. Hegreness M, Shoresh N, Hartl D, Kishony R: An equivalence
principle for the incorporation of favorable mutations in
asexual populations. Science 2006, 311:1615-1617.
43. Colegrave N: Sex releases the speed limit on evolution. Nature
2002, 420:664-666.
Genome Biology 2007, 8:201
http://genomebiology.com/2007/8/1/201
Genome Biology 2007,
44. Imhof M, Schlotterer C: Fitness effects of advantageous mutations in evolving Escherichia coli populations. Proc Natl Acad Sci
USA 2001, 98:1113-1117.
45. de Visser JA, Rozen DE: Clonal interference and the periodic
selection of new beneficial mutations in Escherichia coli.
Genetics 2006, 172:2093-2100.
46. Mukai T, Chigusa SI, Crow JF, Mettler LE: Mutation rate and
dominance of genes affecting viability in Drosophila
melanogaster. Genetics 1972, 72:335-355.
47. Denver DR, Morris K, Lynch M, Thomas WK: High mutation rate
and predominance of insertions in the Caenorhabditis
elegans nuclear genome. Nature 2004, 430:679-682.
48. Denver DR, Morris K, Lynch M, Vassilieva LL, Thomas WK: High
direct estimate of the mutation rate in the mitochondrial
genome of Caenorhabditis elegans. Science 2000, 289:2342-2344.
49. de la Pena M, Elena SF, Moya A: Effect of deleterious mutationaccumulation on the fitness of RNA bacteriophage MS2. Evolution Int J Org Evolution 2000, 54:686-691.
50. Green RE, Krause J, Ptak SE, Briggs AW, Ronan MT, Simons JF, Du L,
Egholm M, Rothberg JM, Paunovic M, et al.: Analysis of one million
base pairs of Neanderthal DNA. Nature 2006, 444:330-336.
51. Kellis M, Birren BW, Lander ES: Proof and evolutionary analysis
of ancient genome duplication in the yeast Saccharomyces
cerevisiae. Nature 2004, 428:617-624.
Genome Biology 2007, 8:201
Volume 8, Issue 1, Article 201
Hegreness and Kishony 201.5